Adult podocytes are terminally differentiated glomerular epithelial cells without regeneration capacity by self-proliferation. Thus, in states of podocyte depletion, rebuilding the kidney at the level of the glomerular filtration barrier is dependent on podocyte regeneration from stem/progenitor cells. Cells of renin lineage (CoRL) and glomerular parietal epithelial cells (PECs) are two recently identified resident progenitors fo adult podocytes in states of podocyte depletion. In this grant proposal we will fully characterize and evaluate their role and function as adult podocyte progenitors, using our bioengineered 3D flow directed microphysiological system (3D MPS) of the glomerular filtration barrier, initially constituting murine podocytes, matrix and endothelial cells. Specific aim (SA)#1 will characterize and evaluate CoRL and PEC progenitor differentiation and biological functions. SA#2 will optimize reprogramming and plasticity of CoRL and PEC progenitors towards a podocyte fate. SA#3 will broaden and validate the scope of podocyte regeneration by additional stem/progenitor cell types such as induced pluripotent stem cells and mesenchymal stem cells. In addition to the availability of cultured CoRL and PECs progenitors, expected deliverables upon completion of the UH2 includes a unique 3D MPS designed to characterize and evaluate CoRL and PEC progenitor viability, differentiation, phenotype, migration and biological function, using several functional assays. Available progenitor cells for study will initially be of mouse origin, taking advantage of their permanent labeling, as they are derived from cell specific reporter mice. The stemness of other candidate podocyte and/or endothelial cell progenitors can also be tested in this 3D MPS by the consortium. Major deliverables for the UH3 are CoRL and PECs that have been reprogrammed to an enhanced podocyte fate through genetic manipulation, changes to the microenvironment, and exposure to specific therapeutics and growth factors. The 3D MPS will be humanized by replacing mouse cells with human cells, and validation of these in vitro studies will be conducted in vivo in cell specific reporter mice where two cell types of interest are simultaneously labeled. Taken together, this grant will provide novel opportunities for the consortia and others to rebuild the kidney at the level of podocytes by resident and non-resident stem/progenitor cells.